Bisphosphonic acids are a group of compounds having a P—C—P skeleton, have high affinity for bone tissues and, when incorporated into osteoclasts, show cytotoxicity and suppress bone resorption. Utilizing such properties for therapeutic purposes, they are used as prophylactic or therapeutic drugs for diseases relating to the fragility of bone such as osteoporosis, osteitis deformans, osteogenesis imperfecta and the like. In addition, bisphosphonic acids containing a nitrogen atom in the side chain such as pamidronic acid, alendronic acid, risedronic acid, zoledronic acid and the like are commercially available as drugs for hypercalcemia of malignancy. Recently, moreover, it has been reported that the disease-free survival is significantly extended when zoledronic acid is used as an adjuvant therapy drug in the endocrine therapy and chemotherapy of premenopausal estrogen sensitive early breast cancer cases and multiple myeloma (non-patent documents 1, 2). Accordingly, the relationship between bisphosphonic acid having a nitrogen atom in the side chain and an antitumor effect is suggested, and direct cytotoxicity and/or indirect cytotoxicity via activation of immunocytes on tumor cells are/is considered to be the cause thereof, though a consensus has not been reached regarding the action mechanism thereof.
For example, a part of zoledronic acid administered to a living body enters into the cell by fluid phase endocytosis, is transferred to nucleoside monophosphate, is converted to a nucleoside triphosphate analog compound, and may antagonistically inhibit biological enzyme reaction utilizing high energy phosphate bond of nucleoside triphosphate, which is suggested to injure tumor cells.
Furthermore, intracellularly transferred bisphosphonic acid has been shown to inhibit farnesyl diphosphate synthase involved in the biosynthesis of isoprenoid metabolites such as cholesterol and the like. The enzyme catalyzes a reaction to synthesize geranyl diphosphate from isopentenyl diphosphate and dimethylallyl diphosphate, and a reaction to synthesize farnesyl diphosphate from isopentenyl diphosphate and geranyl diphosphate. Therefore, inhibition of farnesyl diphosphate synthase interferes with the metabolic pathway downstream of geranyl diphosphate, as well as causes accumulation of isopentenyl diphosphate in the cytoplasm. When the biosynthetic pathway downstream of geranyl diphosphate is inhibited, isoprenoid compounds such as cholesterol, liposoluble vitamins, bile acid, lipoprotein and the like are not biosynthesized, and the proliferation of tumor cells is considered to be suppressed.
The isoprenyl group of farnesyl diphosphate and geranylgeranyl diphosphate biosynthesized by farnesyl diphosphate synthase is transferred to, what is called, small G proteins such as Ras, Rho, Rap, Rab, Rac and the like. The small G protein having the transferred isoprenyl group is translocated to a cellular membrane since the isoprenyl group acts as an anchor, and plays an important role in the proliferation, adhesion and the like of cells. In this case, when nitrogen-containing bisphosphonic acids such as zoledronic acid and the like inhibits farnesyl diphosphate synthase, transfer of the isopropenyl group is inhibited, translocation to the membrane of small G protein is prevented, and, as a result, tumor cell proliferation is inhibited.
When farnesyl diphosphate synthase is inhibited, the intracellular concentration of isopentenyl diphosphate as a substrate thereof increases. The increase in the intracellular concentration of isopentenyl diphosphate is detected by a butyrophilin 3A1 transmembrane protein, and the change thereof is recognized by γδ T cells having a Vγ2Vδ2 T cell receptor. As a result, the γδ T cells are degranulated to release perforin and granzyme B, which induce apoptosis of tumor cells and virus infected cells. It is shown that nitrogen-containing bisphosphonic acids efficiently kill tumor cells and virus-infected cells indirectly via the activation of immunocytes.
The direct and indirect toxicity on tumor cells and virus infected cells as mentioned above by the nitrogen-containing bisphosphonic acids is not effectively induced unless the nitrogen-containing bisphosphonic acids enters into the target cells. However, since bisphosphonic acids clinically applicable at present have all been designed and synthesized for the purpose of improving bone-related diseases, their permeability into tumor cells and virus infected cells is markedly low. In fact, the molecules are negatively charged due to an acid structure of P—C—P it has, thus resulting in markedly low permeability into cells.
While it is disclosed that a pivaloyloxymethyl (POM) group ester derivative of bisphosphonic acid having an unsubstituted 2-pyridyl group, specifically, 2-(pyridyl-2-amino)ethylidene-1,1-bisphosphonic acid tetrakispivaloyloxy methyl ester shows cytotoxicity, and suppresses proliferation of tumor cells, the mechanism thereof has not been clarified (non-patent document 3).
In addition, while bisphosphonic acid derivative can be used by dissolving in DMSO and the like in the in vitro experiment system, when it is administered to the living body, it needs to be solubilized in a biotolerable form. Various techniques have been developed as a means to solubilize medicaments. For example, a method using surfactants such as Tween 80, HC0-60 and the like, a method using clathrating agents such as cyclodextrin (hereinafter sometimes to be referred to as “CD”) and the like, and the like are known. Patent document 1 discloses a production method of alkylated cyclodextrin, and a method of solubilizing hardly water-soluble medicaments by using alkylated cyclodextrin. The alkylated cyclodextrin described in patent document 1 is a methylated cyclodextrin derivative characteristically having an average substitution rate of 1.7-1.9 as measured by 1H-NMR, and the 06-position methylated by 55-75%.